Phosphorylation of P-glycoprotein and its role in multidrug resistance characterization and analysis of adriamycin-resistant J774.2 cells

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Mouse macrophage-like J774.2 cells selected for resistance to either colchicine, vinblastine, or taxol express the multidrug resistance (MDR) phenotype. P-glycoprotein, a family of membrane phosphoglycoproteins, appears to function as an energy-dependent drug efflux pump which maintains a reduced level of drug in MDR cells.;To examine the role of P-glycoprotein phosphorylation in multidrug resistant cells, phosphorylation has been studied in intact cells and in vitro. In vitro experiments have demonstrated that partially purified P-glycoprotein is a substrate for both protein kinase A and protein kinase C. The putative location of one phosphorylated domain in P-glycoprotein has been determined with a site-directed polyclonal antibody.;To analyze the functional aspect of P-glycoprotein phosphorylation, site-directed mutagenesis of the single protein kinase A consensus phosphorylation site in mdr1b cDNA was performed utilizing the polymerase chain reaction. Drug accumulation and efflux experiments with individual clones demonstrated that cells expressing mutated P-glycoprotein effluxed drugs less efficiently than cells expressing wild type P-glycoprotein.;Several MDR variants of J774.2 cells selected with the antitumor drugs taxol, colchicine and vinblastine, all of which interact with the tubulin-microtubule system, had been isolated. It was of interest to develop a resistant cell line selected with a drug that has a different target in the cell. J774.2 cells were made resistant to adriamycin which is known to interact with DNA and inhibit DNA and RNA synthesis.;It is known that there are three mdr genes expressed in rodents: mdr1a, mdr1b and mdr2. Southern blot analysis with an mdr probe revealed that J7.A1-4 expressed all three mdr genes, each of which was amplified. mdr mRNAs were also analyzed by gene specific probes and it was shown that this cell line expressed increased levels of all three mdr mRNAs. However, only the mdr1b type P-glycoprotein was detected by site-directed polyclonal antibodies. The most interesting aspect of this cell line is that it is not most resistant to the selecting drug, adriamycin (129-fold resistance), but it is more resistant to vinblastine (200-fold resistance) and colchicine (172-fold resistance). Drug accumulation of ({dollar}\sp{lcub}14{rcub}{dollar}C) adriamycin and ({dollar}\sp3{dollar}H) vinblastine demonstrated that J7.A1-4 cells can accumulate less ({dollar}\sp3{dollar}H) vinblastine than ({dollar}\sp{lcub}14{rcub}{dollar}C) adriamycin. This further supports the results obtained in cross-resistance experiments. (Abstract shortened with permission of author.).